Stabilisation and incorporation of membrane proteins in semi-permeable separation membranes using amphiphilic heteropolymers

Abstract

The present disclosure provides a semi-permeable membrane comprising an assembly comprising an amphiphilic heteropolymer, a membrane protein and a polyamide thin film composite layer at least partially on top of the amphiphilic heteropolymer-stabilized membrane protein. Methods of forming the semi-permeable membrane are also provided, including stabilizing a membrane protein with an amphiphilic heteropolymer and incorporating the resulting assembly onto a porous support. A polyamide layer may be formed on the membrane by interfacial polymerisation. The amphiphilic heteropolymer-stabilized membrane protein assembly may be immobilized on the support by intermediary of a polymer layer formed by a self-polymerizable compound.

Description

[0001] TITLE

[0002] Stabilisation and incorporation of membrane proteins in semi- permeable separation membranes using amphiphilic heteropolymers .

[0003] TECHNICAL FIELD

[0004] The disclosure relates to a semi -permeable membrane comprising an amphiphilic heteropolymer-membrane protein assembly and methods of making the same .

[0005] BACKGROUND

[0006] Membrane proteins play crucial roles in numerous biological processes , including cellular signalling , transport , and energy production . However, studying and manipulating these proteins outside their native membrane environment presents significant challenges due to their hydrophobic nature and tendency to aggregate or denature in aqueous solutions . Traditional approaches to solubilising membrane proteins have relied on detergents , which can destabilise protein structure and function .

[0007] In recent years , amphipathic polymers known as amphiphilic heteropolymers and amphipols have emerged as promising alternatives to detergents for maintaining membrane proteins in solution . These polymers are designed to wrap around the entire protein, shielding the protein hydrophobic regions from the aqueous environment while preserving the protein' s native structure and function . Amphiphilic heteropolymers have shown potential in various applications , including protein stabilisation, structural studies , and functional assays .

[0008] Despite the advantageous transport processivity and selectivity of transmembrane proteins , their integration into complex synthetic systems remains challenging . Conventional methods for incorporating proteins into separation membranes often involve harsh membrane manufacturing conditions that can compromise protein integrity . Additionally, ensuring proper functionality of membrane proteins within artificial membrane structures continues to be a significant hurdle in the field .

[0009] The development of reverse osmosis ( RO ) and forward osmosis ( FO ) separation membranes with enhanced water permeability and selectivity is an ongoing area of research in water purification technology . While progress has been made in improving membrane materials and structures , the incorporation of biomolecules , such as water channel proteins and solute membrane protein transporters , into RO and FO membranes in a stable and active form has proven difficult . Current methods for protein integration often result in loss of protein function or inadequate incorporation into the membrane matrix , such as unkownn location within the membrane architecture resulting in suboptimal membrane performance and even loss of membrane coating integrity .

[0010] Prior art documents have addressed some aspects of incorporating membrane proteins into synthetic membranes . For example , EP3413998B1 discloses separation membranes comprising selfassembled nanostructures formed between a polyalkyleneimine ( PAI ) , such as polyethyleneimine ( PEI ) , and detergent-stabilised aquaporin water channels . The nanostructures are incorporated into the active layer of the membrane , which is a thin film composite layer formed through interfacial polymerisation . The membranes can be used in hollow fiber modules for applications like forward osmosis , reverse osmosis , and water purification . However , the method may not fully overcome the limitations associated with protein stability and functionality in synthetic membrane environments .

[0011] Another relevant prior art document , W02020115083A1 , relates to amphiphilic vinyl polymers that can solubilise and stabilise membrane proteins in aqueous solution . These polymers contain cyclic hydrophobic groups and form complexes with membrane proteins that maintain the proteins in their native conformation in an aqueous environment . The polymer-protein complexes allow for improved purification, characterisation, and biotechnological applications of membrane proteins compared to existing methods .

[0012] These prior art documents highlight the ongoing efforts to solubilise and stabilise membrane proteins as well as incorporating them on semi -permeable separation membranes . Another known method involves the reconstitution of transmembrane proteins into liposomes or synthetic vesicles . However , this method also presents challenges associated with reconstitution efficiency and vesicle stability at the aqueous-organic interphase to which they may be exposed during subsequent membrane manufacturing steps .

[0013] Thus , there is still room for improvement in developing methods that can effectively stabilise and integrate membrane proteins into semi -permeable separation membranes while maintaining their native structure and the performance properties of the separation membrane . PEI-transmembrane proteins complexes , for instance , are prone to precipitation on the porous support , which greatly hinders the integrity of the polyamide layer obtained by interfacial polymerisation and membrane performance . PEI is a hydrophilic polymer with a very large positive charge density that difficulty caters to the hydrophobic regions of the membrane protein . Therefore , the application of such methods to membrane proteins in complex systems , such as reverse osmosis membranes for water purification, may require further innovation to overcome the challenges associated with protein stability, and functionality in these specialised environments .

[0014] It has , thus , been appreciated that a method for stabilising and incorporating membrane proteins into separation membranes is needed that overcomes one or more of these problems . SUMMARY OF THE INVENTION

[0015] An obj ect of the present invention is to provide a semi-permeable membrane with increased protein integration .

[0016] A further obj ect of the present invention is to provide a semi- permeable membrane with improved performance characteristics , such as water permeability, flux , enhanced salt rej ection and / or enhanced ion selectivity .

[0017] Yet a further obj ect of the present invention is to provide a multi-functional membrane that incorporates various types of amphiphilic heteropolymer-stabilized membrane proteins simultaneously .

[0018] Another obj ect of the present invention is to provide a method for incorporating membrane proteins into semi-permeable membranes by maintaining their solubility and stability .

[0019] A further obj ect of the present invention is to enable the integration of membrane proteins into membrane fabrication processes that may otherwise be detrimental to protein stability, such as interfacial polymerisation .

[0020] Yet another obj ect of the present invention is to overcome challenges associated with protein charging , stability, and functionality when incorporating membrane proteins into semi- permeable membranes comprising a polyamide layer .

[0021] A further obj ect of the present invention is to provide a method for controlled and reproducible immobilization of membrane proteins on porous supports .

[0022] Yet a further obj ect of the present invention is to optimize the amount of amphiphilic heteropolymer-stabilized membrane protein present on the membrane to enhance performance while ensuring reproducibility in membrane production .

[0023] In a first aspect , a semi -permeable membrane is provided . The semi- permeable membrane comprises an assembly comprising an amphiphilic heteropolymer and a membrane protein . This assembly of an amphiphilic heteropolymer and a membrane protein allows for the stabilization of the membrane protein within the semi -permeable membrane structure ; the low free energy of the assembly has strong stabilizing effect which is enough to keep it intact in the membrane coating process . Thereby, the incorporation of functional membrane proteins into semi -permeable membranes while maintaining their stability, i . e . , their structural conformation, and activity is potentially improved . Thus , the presence of amphiphilic heteropolymer-stabilized membrane proteins on a semi -permeable membrane may offer several advantages : The use of amphiphilic heteropolymers provides effective shielding of the hydrophobic regions of the membrane protein . This stabilization may help maintain the protein ' s native structure and function, potentially leading to improved membrane performance over time . By maintaining the proteins in a near-native state , amphiphilic heteropolymer stabilization may help preserve the specific functions of the transmembrane proteins . For example , water channel proteins may retain their ability to facilitate rapid water transport across the membrane .

[0024] Also , by forming an amphiphilic heteropolymer-membrane protein assembly (hereinafter AHMP ) amphiphilic heteropolymers enable membrane protein solubilisation in an aqueous environment .

[0025] Increased solubilisation and stabilisation avoids prevalent aggregation and precipitation of the protein on the support membrane during membrane manufacturing , leading to a polyamide layer ( PA) of better quality . Further , decreased aggregation and precipitation may result in a more uniform distribution of functional proteins across the membrane , potentially leading to more consistent and efficient performance . Also , decreased aggregation and precipitation may increase reproducibility in membrane production . Further, the use of amphiphilic heteropolymers may allow for higher concentrations of functional membrane proteins to be incorporated into the membrane structure . This increased protein density may result in enhanced membrane properties , such as improved water flux , permeability, rej ection or ion selectivity . Thus , this configuration may enable increased protein integration onto the membrane structure and to a semi -permeable membrane with increased salt rej ection .

[0026] In a possible implementation form of the first aspect , the water permeability of said semi -permeable membrane is at least 18L / (m2 * h *bar ) and salt rej ection is at least 99% at 500ppm NaCl , 4 . 9 bar , 25 ° C, pH 6-8 . Specifying the water permanbility and salt rej ection parameters provides a quantitative measure of the membrane ' s performance , allowing for standardised comparison with other membrane technologies and other membrane manufacturing methods .

[0027] In a possible implementation form of the first aspect , the AHMP is immobilised on a porous support . Immobilisation on a porous support enhances the structural integrity of the membrane and provides a stable foundation for the AHMP, potentially improving the overall durability and efficiency of the membrane protein .

[0028] Further, attempts to incorporate a membrane protein into a semi- permeable membrane comprising a PA layer have , to date , been performed by incorporating the membrane protein into the PA layer . This approach brings on challenges associated with protein charging on the porous support , stability, and functionality . AHMP immobilisation on the porous support may advantageously increase the yield of functional protein that is effectively charged on the porous support .

[0029] The use of AHMPs may allow for increased compatibility with membrane fabrication processes : Indeed, the use of amphiphilic heteropolymer and amphipols for obtaining AHMPs may allow for their incorporation into membranes using methods that may otherwise be detrimental to protein stability, such as interfacial polymerisation . In addition, the use of amphiphilic polymers may enable protein incorporation onto membranes that might otherwise be detrimental to the membranes ; previous attempts to incorporate large amounts of protein into PA containing RO membranes have been detrimental to the quality of the PA layer .

[0030] Further, the location of the protein may be ensured, namely on the porous support surface and in the pores of the porous support . Without wanting to be bound by theory, it is currently believed that while the semi -permeable membrane is in operation, this AHMP location may be ideal for drawing an aqueous solution to the environment immediately neighboring the porous support surface . This configuration may result in a net flow of aqueous solution towards the porous support . It was , thus , found by the inventors that flux and permeability of the semi -permeable membrane were surprisingly increased .

[0031] In a possible implementation form of the first aspect , the AHMP is immobilised to the porous support through covalent linking of one or more membrane protein amino acid ( s ) . Covalent linking ensures a strong and stable attachment of the membrane protein to the support , reducing the ris k of protein loss during subsequent membrane manufacturing steps , such as PA layer formation .

[0032] In a possible implementation form of the first aspect , the composition of features of the invention are chosen to obtain a specific type of semi -permeable membrane , such as a reverse osmosis membrane , a forward osmosis membrane or an ultrafiltration or nanofiltration membrane . In short , RO membrane supports are designed for mechanical robustness and pressure tolerance , often at the cost of higher internal transport resistance . FO membrane supports , by contrast , are engineered for mass-transfer efficiency and minimal internal concentration polarization, emphasizing high porosity, low tortuosity, and hydrophilicity over strength . Thus, in a possible implementation form of the first aspect, the porous support composition is varied. The porous support may comprise components selected from a group consisting of poly (acrylonitrile ) (PAN) , poly(vinyl alcohol) (PVA) , poly(vinyl fluoride) (PVDF) , chitosan, polysulfone (PSf) , polyethersulfone (PES) , poly(methyl methacrylate) (PMMA) and poly(vinyl butyral) (PVB) . Tailoring the support layer composition, such as using different porous materials, and combined with AHMP selection and functionality, can allow for production of semi-permeable membranes with distinct performance characteristics .

[0033] In a possible implementation form of the first aspect, the porous support further comprises a second layer formed by selfpolymerisation or self-oligomerisation of a self-polymerisable or a self-oligomerisable compound. The inclusion of said second layer, e.g. , directly on the surface of the porous support may offer additional sites for interaction with the AHMP, potentially increasing the density and uniformity of protein attachment across the membrane surface .

[0034] By the self-polymerisable or self-oligomerisable spontaneous polimerization, an uninterrupted and stable layer or matrix may be formed, which may contribute to more even membrane performance characteristics across the membrane which may be due to the inherent properties of the resulting matrix and / or to more even protein distribution.

[0035] Also, self-oligomerisation and self-polymerisation may result in a stable layer being formed on the porous support. In contrast, a non-stable layer may be produced by use of a non-self- oligomerisable or non-self-polymerisable compound.

[0036] Further,

[0037] In a possible implementation form of the first aspect, the self- polymerisable or self-oligomerisable compound immobilises the AHMP on the porous support by physical adsorption. In a possible implementation form of the first aspect , the self- polymerisable or self-oligomerisable compound is a compound comprising catechol groups with benzenediol rings , such as L-3 , 4- dihydroxyphenylalanine ( L-DOPA) . The compound comprising catechol groups with benzenediol rings may immobilise the AHMP on the porous support by physical adsorption . Without wanting to be bound by theory, it is believed that at least benzenediol rings of the available catechol groups presented by the polymerised L-DOPA interact with aromatic ring side-chains of the membrane protein amino acids by forming attractive , non-covalent pi interactions (by orbital overlap ) .

[0038] In a possible implementation form of the first aspect , the selfoligomerising or a self-polymerising compound may be selected from a group comprising poly-levodopamine (poly-L-DOPA) , polydopamine ( PDA) , polyepinephrine , or polynorepinephrine . In a possible implementation form of the first aspect , a self-polymerisable or self-oligomerisable compound is a combination thereof .

[0039] In a possible implementation form of the first aspect , a self- polymerisable or self-oligomerisable compound forms a matrix .

[0040] In a possible implementation form of the first aspect , said matrix comprises at least some of the AHMP present on the membrane . By forming a matrix, the AHMP may be immobilised by the polymeric compound by entrapment . For this to occur , the AHMP may be firstly mixed with the self-polymerisable or self-oligomerisable compound and then the mix is applied to the porous support . As a result , the self-oligomerisation or self-polymerisation reaction will occur in the presence of the AHMP , potentially reacting with reactive end-groups , such as amines , present on the membrane protein to covalently integrate the AHMP in the forming matrix . By j ointly applying the AHMP and the self-polymerisable or self- oligomerisable compound, process steps can be reduced to a minimum and process efficiency optimised . In a possible implementation form of the first aspect , the polymeric compound is poly-levodopamine , i . e . , a polymer of L-3 , 4 -dihydroxyphenylalanine ( L-DOPA) .

[0041] L-DOPA, also known as levodopa, is a catecholamine precursor that may play a significant role in the functionalisation of the porous support and the immobilisation of amphiphilic heteropolymer- stabilised membrane proteins .

[0042] This poly-levodopamine layer may provide several advantages in the context of the invention, such as enhanced surface functionality : The poly-levodopamine coating may introduce functional groups to the porous support surface , which may increase the number of sites available for protein immobilisation .

[0043] A further advantage may be found in that the catechol and amine groups of poly-levodopamine may enhance the hydrophilicity of the support , which may contribute to improved water flux through the membrane .

[0044] Finally, the exposed reactive groups of poly-levodopamine may allow for various chemical modifications , enabling fine-tuning of the surface properties .

[0045] In a possible implementation form of the first aspect , a matrixlayer is firstly applied to the porous support , and the AHMP is subsequently added to the surface of the formed matrix . In such a case , when the AHMP is applied, most self-polymerization activity may have ceased and EDC-NHS chemistry, or another cross-linking chemistry, may be advantageous for sufficient covalent immobilisation of amphiphilic heteropolymer-stabilised membrane proteins to the porous support via tethering to the matrix .

[0046] EDC-NHS mediated coupling may offer several advantages , such as that the coupling reaction may occur under physiological conditions , potentially preserving the structure and function of the membrane proteins . Also , EDC-NHS chemistry may be a high- efficiency reaction, providing high coupling yields , which may potentially increase the amount and homogeneity in distribution of immobilised protein .

[0047] Thus , the use of L-DOPA in combination with EDC-NHS chemistry may provide a versatile and efficient method for immobilising AHMPs onto porous supports .

[0048] This approach may address challenges associated with protein stability, and / or functionality in semi-permeable membrane environments , potentially leading to improved performance in water purification and solute concentration applications .

[0049] In a possible implementation form of the first aspect , the covalent link is achieved by EDC-NHS chemistry . EDC-NHS chemistry provides a well-established and efficient method for forming covalent links , allowing for controlled and reproducible immobilisation of the membrane proteins .

[0050] In a possible implementation form of the first aspect , at least some of the polyamide thin film composite layer is formed on top of the AHMP, i . e . , such that it is coating the amphiphilic heteropolymer-stabilised membrane protein that is immobilised on a porous support . It was found by the inventors that flux and permeability of the semi -permeable membrane were surprisingly increased by this configuration .

[0051] Without wanting to be bound by theory, it is believed that this configuration allows for the formation of an intact and fully functional PA layer . Because the AHMPs are already present on the surface and in the pores of the porous support during interfacial polymerisation of the PA layer , they may interfere less with the reaction between amines and acyl halides , potentially enhancing the membrane ' s performance by combining the benefits of both the proteins and the polyamide thin film.

[0052] In a possible implementation form of the first aspect , the membrane protein is a membrane water channel or a membrane ion channel , a membrane carrier or a membrane transporter . The use of specific types of membrane proteins allows for tailored functionality of the membrane , such as enhanced water permeability or selective ion transport , depending on the chosen protein . Further, a potential for multi-functional membranes is created : The ability to simultaneously incorporate various types of amphiphilic heteropolymer-stabilized membrane proteins may allow for the creation of membranes with multiple functionalities . For example , a membrane may combine enhanced water permeability with specific ion selectivity or contaminant removal capabilities .

[0053] In a possible implementation form of the first aspect , the average amount of AHMP that is immobilised on the porous support is controlled . Specifying the average density range of immobilised membrane proteins on the semi-permeable membrane allows for optimised performance and reproducibility in membrane production . Performance optimisation refers to an amount of AHMP above which an improvement in membrane performance is not observed or considered no longer cost efficient .

[0054] In a possible implementation form of the first aspect , the AHMP is fluorescently labelled via labelling of the membrane protein . The membrane protein may be labelled with a small-molecule , e . g . , an ATTO dye .

[0055] In a possible implementation form of the first aspect , the amphiphilic heteropolymer used for stabilising the membrane protein is an amphipol .

[0056] In all implementation forms of the first aspect , the amphiphilic heteropolymer or amphipol according to the invention is not an amphiphilic block co-polymer .

[0057] In a possible implementation form of the first aspect the amphiphilic heteropolymer or amphipol used for stabilising the membrane protein may be tethered to or comprise a block copolymer .

[0058] In a possible implementation form of the first aspect , the amphiphilic heteropolymers or amphipols used for stabilising the membrane protein are modified in their chemical composition . Modifying the amphiphilic heteropolymer or amphipol composition, such as using sulfonated or phosphorylated amphipols , can potentially enhance the stability of the membrane protein under specific conditions .

[0059] In a possible implementation form of the first aspect , the method of tethering the AHMP assembly to the matrix is varied . Exploring different tethering methods , such as various chemical crosslinking techniques , can potentially increase the stability and functionality of the immobilised proteins within the membrane structure . Also , more efficient and less costly options may be chosen, such that waste and production time are reduced, or quality may be optimised . Other viable chemistries include azide-alkyne cycloaddition, thiol-ene / thiol-yne click reaction, epoxide coupling , maleimide activated coupling / cross-linking, glutaraldehyde coupling / crosslinking, sulf osuccinimidyl-4- (N- maleimidomethyl ) cyclohexane- 1- carboxylate coupling / crosslinking, imidoester coupling / crosslinking , Michael addition ( including both aza and thia Michael additions ) , photo crosslinking, isocyanate chemistry, acrylate based crosslinking, reductive amination, and aldol addition / condensation . The choice of tethering chemistry may depend on efficiency and ease of implementation, which reflects on process efficiency .

[0060] In a possible implementation form of the first aspect , the composition of the support layer is modified to optimise interaction with the AHMP complex . Tailoring the support layer composition, such as using different porous materials or surface modifications , can potentially enhance the adhesion and stability of the tethered protein complexes , improving overall membrane performance and / or improving aspects of membrane production .

[0061] In a second aspect , a method of preparing a semi -permeable membrane is provided . The method comprises providing a porous support , stabilising a membrane protein with an amphiphilic heteropolymer to form an AHMP assembly, incorporating the AHMP assembly onto the semi -permeable membrane , and forming a first layer consisting of a polyamide layer on the semi-permeable membrane . This method may allow for the efficient and controlled integration of functional membrane proteins into semi -permeable membranes while maintaining their stability and activity while ensuring overall performance of the membrane by combining the benefits of the incorporated membrane proteins with the selective properties of the polyamide layer .

[0062] In a possible implementation form of the second aspect , the polyamide layer may be formed by interfacial polymerisation of specific di- or tri-amines and molecules with polychloride or polysulf onyl chloride . This approach may allow for fine-tuning of the polyamide layer properties , potentially optimising the membrane ' s performance for specific applications .

[0063] In a possible implementation form of the second aspect , the method may further comprise the step of contacting or layering an self- oligomerizable or self-polymerisable compound on the porous support . This additional layer may form a matrix on a surface of the porous support , providing enhanced stability for the amphiphilic heteropolymer-stabilised membrane protein assembly and may improve the overall membrane structure .

[0064] In a possible implementation form of the second aspect , the self- polymerisable compound is L-3 , 4-dihydroxyphenylalanine ( L-DOPA) .

[0065] In a possible implementation form of the second aspect , at least some of the amphiphilic heteropolymer-stabilised membrane protein assembly may be included in a solution comprising the self- oligomerizable or self-polymerisable compound to be layered . This approach may allow for more uniform distribution of the protein complexes within the membrane structure , potentially leading to improved performance . This approach may, further, allow for more efficient immobilisation of the AHMP on the porous support through entrapment of the AHMP within the matrix formed by oligomerisation or polymerisation . Further , by including at least part of the AHMP in the self-polymerisable or self-oligomerisable monomer solution that is layered on the porous support to form the matrix, the matrix is formed in the presence of the AHMP , potentially reacting with pendant reactive groups on the membrane protein and / or the amphiphillic heteropolymer, thereby further contributing to stabilisation of the AHPM within the forming matrix .

[0066] In a possible implementation form of the second aspect , the self- polymerisable or self-oligomerisable compound may be partly polymerised or oligomerised prior to contacting with the porous support .

[0067] In a possible implementation form of the second aspect , the method further comprises the step of chemically treating the amphiphilic heteropolymer or the membrane protein, prior to forming the AHMP assembly, such that covalent linking between the amphiphilic heteropolymer and the membrane protein is introduced .

[0068] In a possible implementation form of the second aspect , the AHMP may be immobilised through covalent linking to functional groups on the porous support or matrix formed by self-oligomerisation or self-polymerisation of the self-oligomerisable or self- polymerisable compound . This covalent attachment may enhance the stability and longevity of the incorporated proteins within the membrane structure .

[0069] In a possible implementation form of the second aspect , the AHMP assembly may be immobilised through covalent linking of pendant amine groups on the amphiphilic heteropolymer to functional groups on the porous support or self-oligomerisable or self-polymerisable compound . This covalent attachment may enhance the density of the AHMP on the porous support .

[0070] In a possible implementation form of the second aspect , the AHMP may be contacted to the porous support in a specific amount range . This controlled loading may allow for optimisation of the amount of AHMP that is finally present on the membrane thereby optimizing membrane performance and ensuring reproducibility in membrane production .

[0071] In a possible implementation form of the second aspect , the amphiphilic heteropolymer or amphipol used for stabilising the membrane protein may be modified in their chemical composition . This modification may potentially enhance the stability of the membrane protein under specific conditions .

[0072] In a possible implementation form of the second aspect , the method may comprise the additional step of tethering a block co-polymer, an oligomer, a homopolymer or a copolymer to the amphiphilic heteropolymer prior to stabilising a membrane protein with the amphiphilic heteropolymer to form an amphiphilic heteropolymer- stabilised membrane protein assembly (AHMP ) .

[0073] In a possible implementation form of the second aspect the amphiphilic heteropolymer or amphipol used for stabilising the membrane protein may be tethered to or comprise a block copolymer .

[0074] In a possible implementation form of the second aspect , the method of tethering the AHMP to the support layer may be by use of EDC- NHS chemistry .

[0075] In a possible implementation form of the second aspect , at least some of the AHMP is immobilised on the porous support , prior to PA layer formation on the semi-permeable membrane .

[0076] In a possible implementation form of the second aspect , at least some of the PA layer is formed on top of the immobilised AHMP .

[0077] In a possible implementation form of the second aspect , the composition of the support layer may be modified to optimise interaction with the AHMP . Tailoring the support layer composition may potentially enhance the adhesion and stability of the tethered protein complexes , improving overall membrane performance . In a possible implementation form of the second aspect , the membrane protein is switched for another class of membrane protein, such as an ion channel or another water channel or a carrier protein . This variation allows for the adaptation of the membrane to different filtration or separation requirements , potentially expanding the range of applications for the membrane technology .

[0078] In a possible implementation form of the second aspect , two or more types or classes of membrane protein are combined, such that a multi-functional membrane incorporating various types of membrane proteins simultaneously is obtained .

[0079] In a possible implementation form of the second aspect , the membrane protein is further labelled fluorescently . A small molecule , e . g . , ATTO dye may be used for labelling .

[0080] In a possible implementation form of the second aspect , the amphiphillic heteropolymer or amphipol is further labelled fluorescently . A small molecule , e . g . , ATTO dye may be used for labelling .

[0081] BRIEF DESCRIPTION OF FIGURES

[0082] FIG . 1 illustrates chemical structures of two polyacrylate-based amphiphilic polymers , A8 -35 according to an embodiment of the present disclosure and a glucose-based non-ionic homopolymer .

[0083] FIG . 2 illustrates a chemical structure of a THAM based non-ionic amphiphilic heteropolymer, according to an embodiment .

[0084] FIG . 3 illustrates chemical structures of two amphiphilic cycloalkane heteropolymers , according to embodiments of the present disclosure .

[0085] FIG . 4 illustrates chemical structures of two amphiphilic heteropolymers , one of which a phosphorylcholine-based zwitterionic amphipol according to an embodiment . FIG . 5 illustrates a structural formula of a complex organic molecule , according to aspects of the present disclosure .

[0086] FIG . 6 illustrates orthogonal views of three PMAL-B series heteropolymers obtained by modification of alternating copolymers of maleic anhydride and a long-chain olefin of different lengths , according to embodiments of the disclosure .

[0087] Fig . 7 illustrates two Western blot images showing protein band patterns reflecting AHMP retention on a porous support according to methods of the present disclosure .

[0088] FIG . 8 depicts a bar graph comparing performance of different membranes , according to methods of the disclosure .

[0089] FIG . 9 shows a bar graph comparing performance of three membrane preparation methods , according to aspects of the present disclosure .

[0090] FIG . 10 illustrates a series of microscopic images showing results of various experimental conditions , according to aspects of the present disclosure .

[0091] FIG . 11 presents a table showing experimental results for different membrane formulations , according to the prior art .

[0092] DETAILED DESCRIPTION

[0093] The present disclosure provides a semi -permeable membrane incorporating assemblies comprising amphiphilic heteropolymers and membrane proteins . This approach offers a novel method for integrating functional membrane proteins into semi-permeable membranes while maintaining their stability and activity . The use of amphiphilic heteropolymers and amphipols for protein stabilisation allows for effective shielding of the hydrophobic regions of the membrane proteins , preserving their native structure and function . This stabilisation enables the proteins to remain functional during the formation of a polyamide layer on a porous support by interfacial polymerisation, a process that would typically cause protein denaturation or loss of function . The resulting semi-permeable membrane may exhibit enhanced performance characteristics , such as improved water flux , permeability, salt rej ection and / or ion selectivity, due to the presence of functional membrane proteins . In some cases , the semi-permeable membrane may be used in applications such as water purification and solute concentration .

[0094] As used herein, the term "semi -permeable membrane" refers to a selective barrier that allows certain molecules or ions to pass through while restricting the passage of others . In the context of this invention, the semi -permeable membrane comprises a porous support structure that incorporates amphiphilic heteropolymer- stabilised membrane proteins and may include additional layers such as a polyamide thin film composite layer .

[0095] As used herein, the term "amphiphilic heteropolymer" refers to a polymer composed of two or more different types of monomers that confer both hydrophilic and hydrophobic properties to the polymer . In contrast to block co-polymers , where different monomer types are organized into well-defined hydrophilic or hydrophobic blocks , each composed of a series of the same monomer units , the monomers composing heteropolymers according to this disclosure do not have a clear separation of monomers into distinct blocks , such that monomers are distributed in a less defined manner , and present different configurations , such as random, alternating , gradient and grafted, where some monomers are hydrophilic and others are hydrophobic . These structures , therefore , allow the polymer to interact with both aqueous and hydrophobic environments .

[0096] In contrast , a non-amphiphilic polymer is composed of monomers with similar chemical properties , resulting in a uniform interaction with its environment . The amphiphilic nature of heteropolymers makes them particularly suitable for stabilizing membrane proteins by shielding the protein ' s hydrophobic regions while maintaining solubility in aqueous solutions . In the context of the invention, the amphiphilic heteropolymer used for stabilising the membrane protein is not an amphiphilic block co-polymer .

[0097] As used herein, the term "amphipol" refers to an amphiphilic polymer specifically designed to stabilise membrane proteins in aqueous solutions . Amphipols are characterized by their ability to form stable complexes with membrane proteins , maintaining protein structure and function in the outside of their native membrane environments . Amphipols are typically composed of statistically distributed hydrophilic and hydrophobic regions optimized for membrane protein stabilization . However , in contrast to amphipathic molecules with distinct separations between hydrophilic and hydrophobic regions , the amphiphilic molecules of the present disclosure have less distinct hydrophilic and hydrophobic regions . Thus , in the context of the present disclosure , the amphiphilic heteropolymer used for stabilising membrane proteins requires at least a portion of statistically distributed hydrophilic and hydrophobic monomers optimized for membrane protein stabilization . Accordingly, an amphipol according to the present disclosure is not a block co-polymer . However , said amphiphilic heteropolymer may further be tethered to or comprise additional monomers arranged to form co-polymer blocks .

[0098] In contrast to detergents , which are characterized by a high critical micelle concentration ( CMC ) , amphipols are amphiphilic polymers with a significantly lower CMC . This key difference allows amphipols to form stable complexes with membrane proteins at much lower concentrations than detergents . While detergents can disrupt protein structure and function at concentrations above their CMC , amphipols maintain protein stability even at concentrations well above their CMC . This property makes amphipols particularly suitable for stabilizing membrane proteins in aqueous environments , as they can effectively shield the hydrophobic regions of the protein without the risk of destabilization associated with detergent micelles .

[0099] While an amphipol could be considered a specialized type of amphiphilic heteropolymer, its unique structural features and functional properties distinguish it from general heteropolymers . Amphipols are characterized by their ability to form stable complexes with membrane proteins , making it possible for them to maintain protein structure and function in the absence of detergents . This specific functionality sets amphipols apart from broader classes of amphiphilic heteropolymers , which may not possess the same capability to effectively stabilise membrane proteins in aqueous environments .

[0100] As used herein, the term "stabilized" refers to a membrane protein that is resilient to industrially relevant challenges , including but not limited to changes in pH , temperature , and exposure to solvents . A stabilized membrane protein retains its structural conformation and functional properties under these conditions . The stabilization is achieved through complexation with amphipols , which effectively shield the hydrophobic regions of the protein and maintain its native conformation . In addition, this stabilization enables the membrane protein to remain functional during the formation of a polyamide layer on a porous support by interfacial polymerisation, a process that would typically cause protein denaturation or loss of function . Importantly, the stabilization provided by amphipols allows for the incorporation of the membrane protein into a membrane structure while preserving its ability to perform its intended function, such as facilitating water or ion transport .

[0101] As used herein, the term membrane protein refers to a protein that is associated with a biological membrane in nature , and may either be an integral membrane protein ( or transmembrane protein) , which span the entire biological membrane , or associate with one or the other side of a membrane , such as peripheral membrane proteins , which are transiently associated with the cell membrane . In the context of this invention, non-integral membrane proteins may exhibit various topologies and functionalities :

[0102] 1 . Membrane enzymes may have many activities , such as oxidoreductase , transferase or hydrolase .

[0103] 2 . Cell adhesion molecules allow cells to identify each other and interact . For example , proteins involved in immune response

[0104] 3 . Lipid-anchored proteins : While not spanning the entire membrane , these proteins may be attached to the membrane via lipid modifications .

[0105] 4 . Cell adhesion molecules : These proteins may mediate interactions between cells or between cells and the extracellular matrix .

[0106] 5 . Structural proteins : Some transmembrane proteins may contribute to membrane structure and stability .

[0107] As used herein, the term "transmembrane protein" refers to a protein that , in nature , spans the entire biological membrane , with portions of the protein exposed on both sides of the lipidic membrane . The hydrophobic domain of transmembrane proteins typically consists of one or more alphahelical regions that interact with the hydrophobic interior of the membranes . Hydrophilic domains tend to have more tertiary structure with hydrophilic surfaces , and so face the aqueous cell interior, periplasm or cell exterior . Thus , the transmembrane proteins of the invention have in common that they present distinct hydrophilic and hydrophobic regions that accomodate aqueous and lipidic environments , respectively .

[0108] In the context of this invention, transmembrane proteins may exhibit various topologies and functionalities :

[0109] 1 . Single-pass transmembrane proteins : These proteins may cross the membrane once , with one domain on each side of the membrane . They may be classified as Type I (N-terminus on the extracellular side ) or Type IT (N-terminus on the cytoplasmic side ) .

[0110] 2 . Multi-pass transmembrane proteins : These proteins may cross the membrane multiple times , forming structures such as alpha-helical bundles or beta-barrels . The number of membrane-spanning segments may vary, ranging from two to over twenty in some cases .

[0111] Functionally, transmembrane proteins may serve various roles :

[0112] 1 . Channels and pores : These proteins may facilitate the passive transport ( such as down a concentration gradient ) of specific molecules or ions across the membrane .

[0113] 2 . Transporters or carriers : These proteins may actively move substances across the membrane , both down and up the concentration gradient , often using energy in the form of ATP or electrochemical gradients .

[0114] 3 . Receptors : These proteins may bind specific ligands and transmit signals across the membrane .

[0115] 4 . Enzymes : Some transmembrane proteins may catalyze chemical reactions at the membrane surface .

[0116] 5 . Cell adhesion molecules : These proteins may mediate interactions between cells or between cells and the extracellular matrix .

[0117] 6 . Structural proteins : Some transmembrane proteins may contribute to membrane structure and stability .

[0118] As used herein, the terms "amphiphilic heteropolymer-stabilised membrane protein" and "amphipol-stabilised membrane protein" refer to an assembly formed between an amphiphilic heteropolymer such as an amphipol and a membrane protein, wherein the heteropolymer or amphipol effectively shields the hydrophobic regions of the membrane protein, possibly maintaining its native structure and function in an aqueous environment . This stabilization may allow the membrane protein to remain functional during incorporation into the semi -permeable membrane structure . As used herein, the term "immobilised" refers to the secure attachment of the amphiphilic heteropolymer-stabilised membrane protein to a surface or structure , in this case , a polymer layer on the porous support . Immobilisation may be achieved through various means , including but not limited to covalent bonding, physical adsorption, or entrapment within a matrix, such that some of the protein remains on the membrane after washes and treatment steps carried out during manufacturing .

[0119] As used herein, the term "porous support" refers to a structural component of the semi-permeable membrane that provides mechanical strength and stability while allowing for the passage of certain molecules or ions . The porous support typically consists of a material with a network of pores or channels , such as polysulfone , polyethersulfone , or polyacrylonitrile , and serves as a substrate for the immobilisation of the heteropolymer-stabilised membrane protein and subsequent formation of additional membrane layers .

[0120] As used herein, the term "polyamide thin film composite layer" refers to a thin, selective barrier layer formed on top of a porous support through interfacial polymerisation . This layer typically consists of a cross-linked polyamide network formed by the reaction between an aqueous solution containing a diamine or polyamine and an organic solution containing a diacyl halide or triacyl halide . The polyamide thin film composite layer is responsible for the primary separation properties of the membrane , including water permeability and solute rej ection . In the context of this invention, the polyamide thin film composite layer is formed on top of or around the heteropolymer-stabilized membrane proteins that are immobilized on the porous support , creating a functional semi -permeable membrane that incorporates both synthetic and biological components for enhanced performance .

[0121] As used herein, the term self-polymerisable or self-oligomerisable compound is a molecule that can undergo chemical reactions to form larger molecular structures — such as oligomers or polymers — without the need for an external co-reactant . These compounds contain reactive functional groups ( for example , double bonds , epoxides , or hydroxyl groups ) that can react with compatible groups between identical molecules . Under appropriate conditions ( such as heat , light , or catalysts ) , they spontaneously link together to form repeating units ( oligomers or polymers ) , generating materials with enhanced mechanical , chemical , or barrier properties . Examples include monomers like acrylates , epoxides , or lactones that polymerise through self-initiated reactions .

[0122] According to a first embodiment , the semi-permeable membrane comprises an amphiphilic heteropolymer-stabilised membrane protein, which is immobilised on a porous support . The amphiphilic heteropolymer-stabilised membrane protein may be any membrane protein, such as for instance one or several of a membrane water channel , a membrane ion channel , a membrane carrier , a membrane- anchored enzyme , or a membrane transporter . The immobilisation of the amphiphilic heteropolymer-stabilised membrane protein on the porous support is targeted through covalent linking of one or more membrane protein amino acid ( s ) and the porous support , whereby the covalent linking is via creation of an amide bond . The covalent link may be achieved by EDC-NHS chemistry .

[0123] In a second embodiment , the porous support further comprises a self-polymerised or self-oligomerised compound with which the covalent link is formed . The polymeric compound may be selected from a group comprising but not limited to poly-levodopamine (poly- L-DOPA) , polydopamine ( PDA) , polyepinephrine , or polynorepinephrine or combinations thereof . These compounds offer reactive groups , such as carboxyl groups , which are exposed for interaction with other components of the semi -permeable membrane . Such reaction may result in the formation of a covalent bond between the self-polymerising or self-oligomerising compound and the AHMP . Alternatively, carboxylic acids of the amphipol are activated via, e . g . , EDC-NHS chemistry and bound to the self-polymerised compound present on the surface of the porous support . In such a case , polyetheramine or other amine-based polymers could be used .

[0124] In other cases , the covalent link is not achieved by EDC-NHS chemistry, e . g . , in a third embodiment , although some covalent interactions may still take place between the AHMP and the selfpolymerising or self-oligomerising compound, the formation of a covalent link is not directly targeted . Rather , physical adhesion of the AHMP is achieved by applying the AHMP to the matrix formed by the oligomerisation or polymerisation of the self-polymerising or self-oligomerising compound that is present on a surface of the porous support . The compound may be present on the porous support as a layer .

[0125] In a fourth embodiment , immobilization is achieved by entrapment , by including at least part of the AHMP in the polymerisable or oligomerisable monomer solution that is layered on the porous support to form the matrix .

[0126] The amphiphilic heteropolymer or the membrane protein may also be chemically treated, prior to forming the AHMP assembly, such that covalent linking between the amphiphilic heteropolymer and the membrane protein is introduced . For example , carboxylic acids of the amphipol may be activated via , e . g . , EDC-NHS chemistry and bound to amine groups present on the surface of the membrane protein .

[0127] According to the invention, at least some of the polyamide thin film composite layer is formed on top of , i . e . , covers the amphiphilic heteropolymer-stabilised membrane protein that is immobilised on a porous support . The polyamide thin film composite layer is formed by interfacial polymerisation using a di- or triamine and a di- or triacyl halide molecule . The di- or tri-amine is typically selected from a group formed by O-phenylenediamine (OPD ) , m-phenylenediamine (MPD) , bisphenol A ( BPA) and 1 trihydroxypropane , and the polychloride or polysulf onyl chloride molecule may be selected from trimesoyl chloride (TMC ) or 1 , 5- naphthalene-bisulf onyl chloride .

[0128] The polyamide layer may be formed by interfacial polymerisation using various combinations and ratios of the specified di- or triamines and di- or triacyl halide molecules . The interfacial polymerisation conditions may also be varied by using different ratios of di- and tri-amines , modifying reaction time and temperature , using different organic solvents , adding catalysts or initiators to control polymerisation kinetics , incorporating additives during polymerisation, and varying the reaction time and temperature to influence crosslinking density .

[0129] In some cases , post-polymerisation treatments may be applied to further modify the polyamide layer . These treatments may include thermal annealing , chemical crosslinking with bifunctional reagents , bleaching or surface modification through plasma treatment or grafting of hydrophilic polymers . The interfacial polymerisation process may be adapted for different membrane geometries , such as flat sheet membranes , hollow fiber membranes , or spiral wound modules .

[0130] Further, the amount of AHMP that is immobilised on the porous support may be controlled . The amount of AHMP immobilised on the porous support may be optimised to achieve desired membrane properties and performance characteristics . Also , a lower limit of a concentration range is useful for protein economy while ensuring semi -permeable membrane performance . The amount of AHMP that is immobilised on the porous support may be titrated by measuring the concentration of AHMP in the solution to be applied to the porous support before and after application . The principle of this method is that protein not present in the solution after exposure to the membrane and subsequent recovery from the membrane is titrated onto the membrane . A solution with a predetermined concentration of AHMP is prepared and a sample is taken . The sample is analyzed by SDS PAGE , absorption at 280nm and UV-VIS HPLC SEC . The concentration of protein and purity is assessed with these methods . The solution is then exposed to the membrane and recovered by pipetting or decanting . The recovered solution is then subj ected to the same analysis . Finally, a wash is performed on the membrane , which is also recovered and assessed in the same way . The amount of protein titrated on the membrane is determined by subtracting the residual and wash concentrations from the charge concentration .

[0131] In some cases , the amphipols and amphiphilic heteropolymers , as used in the context of the present disclosure may be varied in their chemical composition . For instance , several types of amphipols may be employed, each with distinct backbones and side chains . These amphipols may include , but are not limited to , those depicted in the provided chemical structures in Figs 1 - 6 . Each of these amphipols has a unique structure , with varying hydrophilic and hydrophobic regions , which influence their ability to stabilise membrane proteins .

[0132] These amphipols and amphiphilic heteropolymers , as used in the context of the present disclosure , share some common traits but also exhibit important variations that can affect their interaction with membrane proteins and their ability to shield hydrophobic regions :

[0133] Common traits include :

[0134] - Amphiphilic nature with hydrophilic and hydrophobic monomers

[0135] - Ability to form stable complexes with membrane proteins

[0136] - Low critical micelle concentration compared to detergents

[0137] Variations and their potential effects :

[0138] 1 . Backbone structure : The heteropolymers shown in Figures 1-6 have different polymer backbones , ranging from simple carbon chains to more complex structures incorporating heteroatoms like sulphur . These variations in backbone structure can affect the flexibility and conformational properties of the amphipol , potentially influencing how it wraps around and stabilizes the membrane protein .

[0139] 2 . Hydrophilic groups : The heteropolymers exhibit different types and distributions of hydrophilic groups , including carboxyl or carboxylate salt groups , hydroxyl groups , and in some cases , sugar- like moieties . The nature and arrangement of these hydrophilic groups can impact the solubility of the heteropolymer-protein assembly and its interaction with the aqueous environment .

[0140] 3 . Hydrophobic regions : The hydrophobic portions of the heteropolymers vary in length, branching , and chemical composition . Some contain long alkyl chains , while others incorporate cyclic or aromatic structures . These differences in hydrophobic regions can affect how the heteropolymer interacts with the hydrophobic surfaces of the membrane protein, potentially influencing the stability and functionality of the AHMP complex .

[0141] 4 . Functional groups : Some heteropolymers , as shown in Figures 2 and 3 , contain additional functional groups such as amide linkages or cyclic structures . These groups can provide additional points of interaction with the membrane protein or the surrounding environment , potentially enhancing stability or allowing for further chemical modifications .

[0142] 5 . Charge distribution : The heteropolymers differ in their charge distribution, with some containing ionic groups like carboxylates or quaternary ammonium moieties . The charge distribution can affect the electrostatic interactions between the heteropolymer and the membrane protein, as well as the overall solubility and behavior of the assembly in different pH environments .

[0143] 6 . Molecular weight and size : The heteropolymers vary in their overall size and molecular weight , as indicated by the different numbers of repeating units in their structures . The size of the heteropolymer can influence its ability to effectively wrap around and stabilise membrane proteins of different sizes and shapes .

[0144] 7 . Flexibility : Cyclic alkanes , for instance , are very flexible molecules as they can undergo different conformational changes due to their cyclic structure comprising single carbon-carbon bonds . Some other examples of highly flexible molecules are siloxane based, due to the uniquely very flexible -Si-O-Si- bond . Aromatics are generally very rigid due to their flat conformation and -C=C- bonds

[0145] These variations in amphiphilic heteropolymer or amphipol structure can be exploited to optimize the stabilization of specific membrane proteins or to tailor the properties of the resulting heteropolymer-protein complexes for particular applications in membrane fabrication . By selecting or designing amphipols with specific structural features , it may be possible to enhance the stability, functionality, and integration of membrane proteins in semi -permeable membranes , potentially leading to improved performance in applications such as water purification and solute concentration .

[0146] In some cases , modified amphipols may be used to stabilise the membrane protein . These modified amphipols may include , but are not limited to , sulfonated amphipols or phosphorylated amphipols . The introduction of these functional groups can alter the interaction between the amphipol and the membrane protein, potentially enhancing the stability of the protein under specific conditions . For instance , sulfonated amphipols may provide increased stability in acidic environments , while phosphorylated amphipols may enhance stability in alkaline conditions .

[0147] In some cases , amphipols with different hydrophobic / hydrophilic ratios may be used to stabilise the membrane protein . The hydrophobic / hydrophilic ratio of an amphipol can significantly influence its interaction with the membrane protein and its ability to shield the protein ' s hydrophobic and hydrophilic regions . By adj usting this ratio , the amphipol can be tailored to stabilise a specific membrane protein or a group of proteins with similar properties .

[0148] In some cases , the amphiphilic heteropolymer or amphipols may be selected based on their ability to maintain the structural stability and functional properties of the membrane protein during the formation of a polyamide layer on a porous support by interfacial polymerisation . This process , which would typically cause protein denaturation or loss of function, can be carried out without compromising the functionality of the membrane protein when it is stabilized by an appropriate amphipol .

[0149] In some cases , the amphipols may be selected based on their ability to form stable complexes with the membrane protein at low concentrations . This property, characterized by a low critical micelle concentration (CMC ) , distinguishes amphipols from detergents and allows them to stabilise membrane proteins effectively at much lower concentrations .

[0150] The amphipols used in the present disclosure may be random heteropolymers . Random heteropolymers are a class of polymers composed of two or more different types of monomers arranged in a random sequence along the polymer chain . Unlike alternating copolymers , which have a regular , repeating pattern of monomers , random heteropolymers exhibit a statistical distribution of monomer units . This random arrangement of monomers can provide unique properties that make them suitable for stabilizing membrane proteins in aqueous environments .

[0151] The amphiphilic nature of random heteropolymers allows them to interact effectively with the hydrophobic regions of membrane proteins while maintaining solubility in aqueous solutions . The random distribution of hydrophilic and hydrophobic units along the polymer chain provides flexibility in adapting to the varied surface properties of different membrane proteins . Further, the heterogeneous composition allows for multiple types of interactions with the protein surface , potentially leading to more stable protein-polymer complexes . The random arrangement of monomers and heterogeneity of polymers can provide a more adaptable interface between the protein and the aqueous environment , accommodating variations in protein structure and surface properties , and compensating for errors in prediction of polymer- protein interaction . Thus , the flexibility in monomer composition and ratio in random heteropolymers allows for complexation of a broader range of membrane proteins in an aqueous environment .

[0152] The amphipols used in the present disclosure may be alternating copolymers , which offer unique advantages for stabilizing membrane proteins . Alternating copolymers are characterized by a regular, alternating sequence of two different hydrophilic and hydrophobic monomer units along the polymer chain .

[0153] PMAL polymer structure is significantly different from that of the the random heteropolymers discussed above : Rather than featuring a random distribution of interspersed hydrophilic and hydrophobic groups , PMAL polymers ( Fig . 6 ) are formed from a succession of pairs of groups , one of which is hydrophobic and the other hydrophilic . The hydrophobic groups bear C8 , C12 , or C16 alkyl chains . The nature of the hydrophilic groups depends on the type of PMAL . In PMAL-B-100 , each polar group is comprised of one carboxylate and one ammoniumamide , as shown in Fig . 6 . In PMAL-B- 0 , it comprises two carboxylates , making the polymer chemically similar to a grafted polyacrylate but for the regular alternation of two carboxylates and one alkyl chain . PMAL-B-50 features a 1 : 1 mixture of these two types of polar groups , presumed to be randomly distributed . PMAL-C8 , PMAL-C12 , and PMAL-C16 are versions of PMAL- B-100 carrying C8 , C12 , and C16 alkyl chains , respectively .

[0154] The regular alternation of hydrophilic and hydrophobic units may allow for more consistent and predictable interactions with membrane proteins and better matching with the amphiphilic nature of membrane proteins , potentially leading to more stable and functional protein-amphipol complexes .

[0155] As is the case for other amphiphilic heteropolymers , alternating copolymer amphipols may be designed with various functional groups incorporated into either the hydrophilic or hydrophobic monomers . This can allow for additional customization of the amphipol ' s properties , such as pH responsiveness , charge distribution, or the ability to form specific interactions with the membrane protein or surrounding environment .

[0156] The amphipols used in the present disclosure may be gradient heteropolymers . Gradient heteropolymers , also known as gradient copolymers , are a type of copolymer where the composition of monomers changes gradually along the length of the polymer chain . Unlike block copolymers with distinct monomer blocks or random copolymers with no specific monomer order , gradient copolymers have a smooth transition in monomer composition along the chain . This gradual change may result in less intrachain and interchain repulsion compared to other copolymers . Gradient copolymers are typically synthesized using controlled radical polymerisation techniques , which may allow precise control over the monomer composition throughout the polymerisation process . The unique structure of gradient copolymers may make them suitable for stabilizing membrane proteins by mimicking the natural lipid environment of cell membranes . Some potential advantages of gradient copolymers include the ability to create a more naturallike environment for membrane proteins , which may help maintain their structure and function . Additionally, gradient copolymers may be tailored to match the specific requirements of different membrane proteins , potentially making them versatile tools for protein stabilization .

[0157] The amphipols used in the present disclosure may be polyacrylate- based amphipols . Polyacrylate-based amphipols represent an important class of amphiphilic polymers used for stabilizing membrane proteins . These amphipols are typically composed of a hydrophilic backbone with hydrophobic side chains , providing a balance of properties that allows them to effectively shield the hydrophobic regions of membrane proteins while maintaining solubility in aqueous environments .

[0158] The chemical composition of polyacrylate-based amphipols generally includes a polyacrylate backbone with various pendant groups . The hydrophobic component often consists of alkyl chains of varying lengths , while the hydrophilic portions may include carboxyl groups , hydroxyl groups , or other polar moieties . This combination of hydrophobic and hydrophilic elements allows the amphipol to wrap around the transmembrane regions of proteins , effectively replacing the lipid bilayer and maintaining protein stability in solution .

[0159] A common example of a polyacrylate-based amphipol is A8-35 ( Fig . 1A) , which consists of a polyacrylate backbone with approximately 35 % of the carboxylates bearing octylamine groups and 25% bearing isopropylamine groups . This structure provides a balance of hydrophobicity and hydrophilicity that has proven effective for stabilizing a wide range of membrane proteins .

[0160] Another example of polyacrylate-based amphipol are PAA modified with cyclic hydrocarbon groups of 8 carbon atoms ( Figs . 3A & 3B ) . Using amphipols with hydrophobic moieties of cyclic nature presents at least the advantage of recovering a higher amount of membrane protein at low polymer concentrations over a short period of incubation, thereby improving membrane protein solubilisation and stabilisation .

[0161] The molecular weight and polydispersity of polyacrylate-based amphipols can be controlled through various polymerisation techniques , allowing for the production of amphipols with specific properties tailored to different applications or protein types . The ratio of hydrophobic to hydrophilic groups can also be adj usted to optimize protein stability and solubility for particular experimental conditions or membrane protein characteristics . The amphipols used in the present disclosure may be phosphoryl choline-based amphipols . Phosphorylcholine-based amphipols represent an important class of amphipathic polymers used for stabilizing membrane proteins . These amphipols are characterized by the presence of phosphorylcholine groups , which mimic the zwitterionic head groups of phosphatidylcholine lipids found in biological membranes . The phosphorylcholine moiety consists of a quaternary ammonium cation and a phosphate anion, providing a neutral overall charge while maintaining high hydrophilicity ( Fig . 4 ) .

[0162] Phosphoryl choline-based amphipols are typically derived from methacrylate or acrylamide monomers , with pendant phosphoryl choline groups . This structure allows the amphipol to interact with the hydrophobic regions of membrane proteins while maintaining excellent solubility in aqueous environments . The zwitterionic nature of the phosphorylcholine groups contributes to the amphipol ' s ability to shield membrane proteins from aggregation and denaturation .

[0163] Compared to other amphipols like A8 -35 , phosphoryl choline-based amphipols offer several advantages . The biomimetic nature of the phosphoryl choline groups may provide a more native-like environment for membrane proteins , potentially enhancing their stability and functionality . Additionally, the zwitterionic character of the phosphoryl choline groups contributes to the amphipol ' s ability to maintain protein stability across a wide range of pH values and ionic strengths . Phosphorylcholine-based amphipols have demonstrated excellent performance in maintaining the structural integrity and activity of various membrane proteins , including G protein-coupled receptors , ion channels , and transporters .

[0164] The amphipols used in the present disclosure may be styrene maleic anhydride ( SMA) , see Fig . 2B . SMA may be considered a unique type of amphipathic polymer that can form synthetic nanodiscs . Nanodiscs may be small , disc-shaped complexes composed of the SMA polymer , phospholipids from cell membranes , and a membrane protein . The formation of these nanodiscs may allow for the stabilization and solubilization of membrane proteins without the need for detergents .

[0165] In some cases , SMA polymers may have a distinctive structure consisting of alternating styrene and maleic anhydride units . This structure may provide both hydrophobic ( styrene ) and hydrophilic (maleic anhydride ) regions , allowing the polymer to interact with both the lipid bilayer and the aqueous environment . The maleic anhydride units may hydrolyze in aqueous solutions , forming carboxyl groups that can further enhance the polymer ' s amphipathic properties .

[0166] The process of nanodisc formation with SMA may involve the polymer interacting with lipid bilayers , potentially extracting membrane proteins along with their surrounding lipids . This extraction process may result in the formation of disc-shaped particles , where the hydrophobic regions of the SMA polymer may interact with the lipid acyl chains , while the hydrophilic regions may face the aqueous environment or cover the hydrophilic heads of the lipids .

[0167] In some cases , the size of the SMA nanodiscs may be controlled by adj usting the polymer-to-lipid ratio or the molecular weight of the SMA polymer . This size control may allow for the optimization of nanodisc formation for different membrane proteins or applications .

[0168] The use of SMA for membrane protein stabilization may offer several potential advantages . Unlike traditional detergent-based methods , SMA may preserve the native lipid environment around the membrane protein, potentially maintaining its structural integrity and functional properties . Additionally, the SMA nanodiscs may be stable in solution for extended periods , possibly facilitating semi -permeable membrane fabrication . Non-ionic amphipols represent another important class of amphipathic polymers for membrane protein stabilization . These amphipols are characterized by their lack of charged groups , which can provide advantages in certain transmembrane protein stabilisation applications . Two main types of non-ionic amphipols have currently been developed : THAM-based amphipols ( Fig . 2A) and glucose-based amphipols , such as hetero- and homo-polymeric glucose-based non-ionic polymers ( Figs . IB & 5 ) .

[0169] THAM-based non-ionic amphipols are derived from tris (hydroxymethyl ) aminomethane (THAM) and typically consist of hydrophilic glucose and trihydroxy amide units and undecane carbamate hydrophobic unit . These amphipols can effectively stabilise membrane proteins while avoiding potential electrostatic interactions that may occur with charged amphipols . The absence of charged groups makes THAM-based amphipols particularly useful in applications where pH changes or high salt concentrations may affect the stability of ionic amphipol-protein complexes .

[0170] Glucose-based non-ionic amphipols , on the other hand, utilize glucose units as their hydrophilic components . These amphipols often feature glucose moieties attached via various linkers . The use of glucose provides excellent water solubility and biocompatibility, making these amphipols most suitable for membrane protein solubilisation . The multiple hydroxyl groups of glucose also offer opportunities for further functionalization, allowing for tailored properties to suit specific membrane proteins or experimental conditions .

[0171] Non-ionic amphipols offer several advantages over their ionic counterparts . Their lack of charged groups reduces the potential for unwanted electrostatic interactions with membrane proteins or other components in the system, such as interfacial polymerisation reactants . This property can be particularly beneficial when working with proteins sensitive to changes in ionic strength or pH . Additionally, non-ionic amphipols may exhibit improved stability in high-salt environments and may be less likely to interfere with protein-protein interactions .

[0172] In the context of the present invention, RAFT polymerisation, or reversible addition-fragmentation chain-transfer polymerisation, may be utilized to synthesize amphiphilic heteropolymers with well- defined molecular weights , narrow molecular weight distributions , and complex architectures . This controlled radical polymerisation technique employs a chain transfer agent (CTA) to mediate the polymerisation process , providing control over polymer growth and enabling the production of polymers with specific end-group functionalities .

[0173] RAFT polymerisation offers several advantages for synthesizing polymers compared to other polymerisation methods . It provides high functionality and high degree of chain length control , allowing for the synthesis of amphiphilic heteropolymers , amphipols ; dendritic, comb, brush, star copolymers and other complex structures . The technique is compatible with a wide range of monomers used in amphiphilic heteropolymer synthesis and can be performed under various reaction conditions , including in aqueous media . Additionally, RAFT polymerisation may result in polymers and amphipols with narrow polydispersity indices , indicating a more uniform molecular weight distribution . The ability to retain the CTA at the polymer chain end may also allow for further functionalization or chain extension of the amphipols .

[0174] In some cases , RAFT polymerisation for amphipol synthesis may have some limitations . The synthesis and purification of RAFT agents suitable for amphipol production can be challenging and timeconsuming . The technique may also be sensitive to impurities and require careful control of reaction conditions to achieve optimal results in amphipol synthesis . In some cases , the presence of the RAFT agent end-group may affect the properties of the final polymer, necessitating post-polymerisation modifications . The transmembrane protein used in the context of the present disclosure may be a membrane water channel . Membrane water channels , some of which are known as aquaporins , are a family of integral membrane proteins that facilitate the transport of water across biological membranes . Aquaporins may be particularly useful in the context of semi -permeable membranes for water purification or desalination, as they can provide highly selective and efficient water transport pathways . Different types of aquaporins may be used, each with distinct water transport properties and selectivities . For instance , aquaporin Z (AqpZ ) is a well-studied water channel protein known for its high water permeability and strict exclusion of ions and other solutes .

[0175] In other cases , the transmembrane protein may be a membrane ion channel . Membrane ion channels are proteins that form pores in biological membranes , allowing for the selective passage of ions across the membrane . These proteins can be selective for specific types of ions , such as sodium, potassium, calcium, or chloride ions , and may be gated by various stimuli , including voltage , ligand binding, or mechanical stress . The use of membrane ion channels in semi -permeable membranes may enhance the selectivity and efficiency of ion transport , potentially improving the performance of the membrane in applications such as ion exchange or electrodialysis .

[0176] In yet other cases , the transmembrane protein may be a membrane carrier or a membrane transporter . These proteins facilitate the transport of specific molecules across biological membranes , with membrane transporters facilitating specific transport against a concentration gradient and membrane carriers facilitating specific transport along a concentration gradient . Membrane carriers and transporters can be selective for a wide range of solutes , including sugars , amino acids , nucleotides , and various ions . The use of these proteins in semi-permeable membranes may enable the selective transport of specific solutes , potentially enhancing the separation performance of the membrane .

[0177] In some cases , other types of membrane proteins may be used in the context of the invention . These may include , but are not limited to , membrane receptors , membrane enzymes , or membrane adhesion molecules . The choice of membrane protein may depend on the specific application of the semi -permeable membrane , the desired transport properties , and the compatibility of the protein with the amphipol and the membrane fabrication process .

[0178] In some cases , the amphiphilic heteropolymer or amphipol-stabilised membrane protein may be immobilised on a polymeric layer on the porous support . The immobilisation process may involve covalent linking of one or more amino acid residues of the membrane protein . This covalent linking may be achieved through various chemical reactions , including but not limited to EDC-NHS chemistry . Other chemistries include azide-alkyne cycloaddition, thiol-ene / thiol- yne click reaction, epoxide coupling, maleimide activated coupling / cross-linking , glutaraldehyde coupling / crosslinking, sulf osuccinimidyl-4- (N-maleimidomethyl ) cyclohexane-l-carboxylate coupling / crosslinking, imidoester coupling / crosslinking , Michael addition ( including both aza and thia Michael additions ) , photo crosslinking , isocyanate chemistry, acrylate based crosslinking , reductive amination, and aldol addition / condensation .

[0179] EDC-NHS chemistry refers to a common method for creating amide bonds between carboxyl and amine groups . In this case , the carboxyl group may be present on the porous support or on a polymeric or oligomeric compound layer on the porous support , and the amine group may be present on the membrane protein .

[0180] The EDC-NHS coupling reaction may proceed as follows :

[0181] 1 . Activation : The carboxylic acid groups on the poly-levodopamine coating or the porous support may be activated by EDC to form an O-acylisourea intermediate . 2. NHS ester formation: NHS may react with the O-acylisourea intermediate to form a stable NHS ester.

[0182] 3. Protein coupling: The NHS ester may then react with primary amine groups on the amphipol-stabilised membrane protein, forming a stable amide bond.

[0183] The resulting covalent bond may securely tether the amphipol- stabilised membrane protein to the polimeric or oligomeric compound, ensuring its stability and functionality during subsequent membrane fabrication steps .

[0184] Indeed, as seen in Fig. 10, EDC-NHS activation is required for efficient AHMP retention on an L-DOPA coated porous support (Figs. 10 (c) vs. 10 (d) . When comparing to when the support is coated instead with polydopamine, this result points to the advantage of the carboxylic acid groups of the poly-L-DOPA for EDC-NHS crosslinking of the AHMP. This result is further supported by the functional result of Fig. 9, where both flux and salt rejection are increased (second bar) in comparison to where no AHMP is tethered to the membrane (third bar) .

[0185] Fig. 9 Further shows how performance is improved where the AHMP is included in the self-polymerisable compound solution to be layered on the membrane, i.e., one-pot approach, third bar, in comparison with where the matrix is firstly formed on the support layer and AHMP is subsequently tethered to it (second bar) .

[0186] In some cases, the porous support may be coated with a selfoligomerising or a self-polymerising compound to form a matrix layer. Said self-oligomerising or a self-polymerising compound may be selected from a group comprising poly-levodopamine (poly- L-DOPA) , polydopamine (PDA) , polyepinephrine, or polynorepinephrine prior to the immobilization of the amphipol- stabilised membrane protein. These compounds offer reactive groups, such as carboxyl groups, which are exposed for interaction with other components of the semi -permeable membrane. Such reaction may result in the formation of a covalent bond between the selfpolymerising or self-oligomerising compound and the AHMP .

[0187] In some cases, the self-polymerisable or self-oligomerisable compound may be chemically modified before being layered on the porous support .

[0188] The self-polymerisable or self-oligomerisable compound may be partly polymerised or oligomerised prior to contacting with the porous support. Prepolymerized molecules that are water soluble and can have a good interaction with a polysulphone (PSf) porous support (carrying either amines or carboxylic acids) are: poly(L- DOPA) , poly ( dopamine ) , poly ( epinephrine ) , poly (norepinephrine) , poly(acrylic acid) , poly (methacrylic acid) , poly ( ethyleneimine ) , poly (allylamine hydrochloride) , poly ( glutamic acid) , poly (aspartic acid) , poly ( vinylamine ) , poly(vinyl alcohol) , poly ( vinylpyrrolidone ) , chitosan, poly ( acrylamide ) , poly(vinyl acetate) , poly (aspartic acid) .

[0189] In the context of the invention, the self-polymerisable or self- oligomerisable compound may be a compound selected from a group including, but not limited to catechols and catecholamines, polyphenols, polysaccharides, thiols, alkoxysilanes, acrylates, quinones, aromatic amines and phenolic compounds. To mention some examples of catechols: Catechol, epinephrine, norepinephrine, 5,6- dihydroxyindole, 5 , 6-dihydroxyindole-2-carboxylic acid and hydroquinone. Examples of polyphenols: Tannic acid, resveratrol, epigallocatechin gallate, quercetin, curcumin, gallic acid, ellagic acid and catechin. Further, the self-polymerisable or self- oligomerisable compound may be chemically modified, such as functionalised with by addition of functional side groups.

[0190] Poly-L-DOPA and PDA are both derived from the self-polymerisation of L-DOPA or dopamine, respectively. These polymers are known for their strong adhesion properties, which, in the context of the invention, can facilitate the immobilization of the protein assembly on the porous support. L-DOPA, also known as levodopa, is a catecholamine precursor that may play a significant role in the functionalisation of the porous support and the immobilisation of amphipol-stabilised membrane proteins .

[0191] L-DOPA contains several reactive groups that may contribute to its functionality in the context of membrane modification . These groups include :

[0192] 1 . Catechol group : The dihydroxyphenyl moiety of L-DOPA may undergo oxidation to form quinones , which can react with various nucleophiles .

[0193] 2 . Amine group : The primary amine of L-DOPA may participate in various reactions , including the formation of amide bonds .

[0194] 3 . Carboxylic acid group : This group may be involved in the formation of ester or amide bonds .

[0195] Both Poly-L-DOPA and PDA are formed through a self-polymerisation process , where the monomer units (L-DOPA or dopamine ) undergo a series of oxidation and cyclization reactions to form a polymer chain . This process results in polymers with a high density of reactive groups , including catechol and amine groups , which can interact with various surfaces and form strong adhesive bonds .

[0196] The reactive groups exposed on the surface of the polymerised L- DOPA or PDA can play a crucial role in protein immobilization . Specifically, the catechol groups can form strong covalent and non- covalent interactions with various functional groups on the protein surface , while the amine groups can participate in covalent bond formation with carboxyl groups on the protein or the porous support . These interactions can effectively tether the protein assembly to the support , ensuring its stability during subsequent membrane fabrication steps .

[0197] Also , the membrane protein may also interact , e . g . , via its amines or thiols . Thiols and amines can participate in covalent interactions via enones or quinones with one of the oxidative forms of L-DOPA, dopamine or other self-polymerizable or self- oligomerisable compounds .

[0198] As seen in Fig . 7 , protein titration onto the membrane surface is observed for polysulfone membranes coated either in L-DOPA or Dopamine . Protein loading is determined by comparing the Coomassie signal intensity in the sample ( titled Control , L-DOPA or PDA) to the ' After Coating (AC ) ' sample and the ' wash after coating ' (WAC ) sample . A weaker signal in the AC sample and WAC sample compared to the original sample indicates that protein has been titrated on the membrane . The control sample was performed on a membrane lacking any coating and showed no protein retention .

[0199] Performing titration on a membrane coated either with L-DOPA or Dopamine resulted in titration, based on the less intense AC sample compared to the original sample .

[0200] While both poly-L-DOPA and PDA can be used for protein immobilization, there are some differences in their exposed functional side groups that may influence their performance .

[0201] Poly-L-DOPA, derived from the self-polymerisation of L-DOPA, retains the carboxyl groups of the L-DOPA monomer, which can provide additional sites for covalent bond formation with the protein . On the other hand, PDA, derived from the selfpolymerisation of dopamine , lacks these carboxyl groups .

[0202] The use of poly-L-DOPA or PDA for protein immobilization offers several advantages : The strong adhesion properties of these polymers can ensure a secure attachment of the protein assembly to the porous support , reducing the ris k of protein loss during membrane fabrication . The presence of multiple reactive groups on the polymer surface can provide multiple points of attachment for the protein, potentially enhancing the stability of the immobilized protein complex . Furthermore , the ability to form covalent bonds with the protein can provide a durable linkage that can withstand the harsh conditions of interfacial polymerisation, ensuring the retention of protein functionality in the final membrane product .

[0203] As seen in Fig . 8 , the formation of a poly-L-DOPA layer in the presence of an AHMP ( comprising the amphipol-stabilised transmembrane protein AqpZ ) outperforms where a layer of poly-L- DOPA only is formed ( in the absence of AqpZ ) in terms of both water flux and salt rej ection . Moreover , it can be aprreciated that this effect cannot be replicated if L-DOPA (which contains a carboxylic group ) is exchanged for dopamine (which does not contain any carboxylic groups ) . This result suggests that the presence of the carboxyl groups of L-DOPA is advantagous for a successful AHMP immobilization by entrapment in the method where the transmembrane protein is included in the solution comprising the polymerisable compound to be layered . This conclusion is further corroborated with the fluorescence images (L-DOPA vs . Dopamine ) of Fig . 10 .

[0204] In some cases , the AHMP may be immobilised on the porous support without the need for covalent crosslinking using additional activators in the method . For instance , the protein assembly may be layered on top of a self-polymerising compound layer, such as a poly-L-DOPA layer , on the porous support . The protein assembly may adhere to the resulting matrix through physical adsorption, forming a stable interface between the protein and the support .

[0205] In other cases , the AHMP may be entrapped within a matrix during its formation on the support . For example , the protein assembly may be added to a solution of self-polymerisable or self- oligomerisable monomer , such as an L-DOPA monomer solution, prior to or during polymerisation . The resulting polymer matrix may encapsulate the protein complex, immobilising it within the structure of the porous support and / or resulting matrix .

[0206] The formation of the polyamide layer on the porous support involves an interfacial polymerisation process . This process may involve the reaction between an aqueous solution containing a diamine or polyamine and an organic solution containing a diacid chloride or triacid chloride . The diamine or polyamine and the diacid chloride or triacid chloride react at the interface between the aqueous and organic phases to form a thin film of cross -linked polyamide on the surface of the porous support . This polyamide layer serves as the selective barrier in the semi-permeable membrane , allowing for the passage of certain molecules or ions while restricting others .

[0207] The choice of diamine or polyamine and diacid chloride or triacid chloride used in the interfacial polymerisation process can be varied to tailor the properties of the resulting polyamide layer . For example , the diamine or polyamine may be selected from a group including, but not limited to , O-phenylenediamine (OPD ) , m- phenylenediamine (MPD) , bisphenol A ( BPA) , and trihydroxypropane . These compounds offer a range of reactivity and cross-linking potential , allowing for the adj ustment of the polyamide layer ' s density, permeability, and mechanical properties .

[0208] Similarly, the diacid chloride or triacid chloride may be selected from a group including , but not limited to , trimesoyl chloride (TMC ) and 1 , 5-naphthalene-bisulf onyl chloride . These compounds provide different degrees of cross-linking and hydrophobicity in the resulting polyamide layer , which can influence the membrane ' s selectivity and water permeability .

[0209] In some cases , alternative monomers may be incorporated into the interfacial polymerisation process to modify the structure and performance of the polyamide layer . These may include other aromatic diamines , aliphatic diamines , polyethyleneimine , alternative acyl chloride monomers , multifunctional monomers , monomers with additional functional groups , and cyclic monomers . The use of these alternative monomers can provide additional options for tuning the properties of the polyamide layer, such as its hydrophilicity, cross-linking density, free volume , and rigidity .

[0210] In some cases , the conditions under which the interfacial polymerisation is carried out can also be varied to optimize the incorporation of the AHMP into the polyamide layer. This may involve adjusting parameters such as the reaction time, temperature, and the ratio of the diamine or polyamine to the diacid chloride or triacid chloride. These adjustments can influence the cross-linking density, layer thickness, and other properties of the resulting polyamide layer, potentially enhancing the performance of the semi-permeable membrane.

[0211] In some cases, the polyamide layer or thin film composite layer formation may involve various monomer reactant alternatives to enhance membrane performance and properties . The conventional monomers used in interfacial polymerisation, such as m- phenylenediamine (MPD) and trimesoyl chloride (TMC) , may be replaced or supplemented with alternative compounds to tailor the membrane characteristics .

[0212] For instance, the aqueous phase monomer MPD may be substituted with other diamines or polyamines. These may include, but are not limited to, piperazine (PIP) , polyethyleneimine (PEI) , or modified aromatic diamines such as 2 , 5-diaminobenzenesulf onic acid. The aqueous phase monomer may also be primary, secondary and tertiary alcohols, polyols, phenols and polyphenols. The use of these alternative monomers may result in changes to the membrane's hydrophilicity, charge density, and pore structure. As an example, the use of PIP in the place of MPD will give rise to an ultrafiltration membrane.

[0213] In the organic phase, TMC may be replaced with other acyl chlorides or sulfonyl chlorides. Examples may include isophthaloyl chloride (IPC) , terephthaloyl chloride (TPC) , or 5-isocyanato-isophthaloyl chloride (ICIC) . These alternatives can affect the degree of crosslinking, membrane thickness, and chemical resistance of the resulting polyamide layer.

[0214] The incorporation of monomers with additional functional groups may further modify membrane properties. For example, monomers containing carboxylic acid, sulfonic acid, or quaternary ammonium groups may alter the membrane ' s surface charge and hydrophilicity . In some cases , the use of monomers with multiple reactive sites , such as dendritic or hyperbranched polymers , may lead to increased crosslinking density and improved selectivity . Indeed, the polyamide layer may be formed by interfacial polymerization using various combinations and ratios of hyperbranched polymers . Hyberbranched polymers ( HBPs ) are macromolecules with a highly branched structure and abundant terminal groups . HBPs may be selected from polyamidoamine ( PAMAM) , polyethyleneimine ( PEI ) , and acyl chloride-terminated hyperbranched polyesters ( HBPACs ) . The interfacial polymerization conditions may also be varied by using different ratios of HBPs , modifying reaction time and temperature , using different organic solvents , and / or adding catalysts or initiators to control polymerization kinetics , incorporating additives during polymerization, and varying the reaction time and temperature to influence crosslinking density .

[0215] Cyclic monomers , such as cyclodextrins or calixarenes , may be employed to create membranes with intrinsic microporosity . These structures can potentially enhance both the permeability and selectivity of the membrane by providing well-defined pathways for molecular transport .

[0216] The ratio of monomers used in the interfacial polymerisation process may be adj usted to fine-tune membrane properties . For instance , varying the ratio of diamine to polyamine in the aqueous phase can influence the membrane ' s charge density and pore size distribution . Similarly, altering the concentration of acyl chloride in the organic phase may affect the degree of crosslinking and membrane thickness .

[0217] In some cases , the use of additives or co-monomers during the interfacial polymerisation process may further modify membrane characteristics . These may include surfactants to control interfacial tension, nanoparticles to enhance permeability or fouling resistance , or small molecules to act as pore-forming agents .

[0218] The reaction conditions , such as temperature , pH , and solvent composition, may be optimized for each specific combination of monomers to achieve desired membrane properties . Postpolymerisation treatments , such as chemical modification or thermal annealing, may be applied to further enhance membrane performance .

[0219] In an alternative approach, the semi -permeable membrane according to this disclosure may not comprise a polyamide or TFC layer . Rather , polyelectrolyte layer by layer deposition may be used to interact with the surface charge of the amphiphilic heteropolymer or amphipol-stabilised membrane protein . Such an aproach can result in a semi-permeable membrane for different separation purposes .

[0220] Polyelectrolyte complexes such as HAUCI4 - 4^0, NaBH poly ( dimethyl diallyl ammonium chloride ) ( PDADMAC ) and / or poly ( sodium-4 - styrenesulf onate ) ( PSS ) can be alternatively deposited (by a layer by layer ( LbL ) deposition method ) on a polymeric membrane surface that is charged with amphiphilic heteropolymer or amphipol- stabilised membrane protein to produce a membrane that can serve as a semi-permeable membrane for different separation purposes . Amphiphilic heteropolymer or amphipol-stabilised membrane protein can also be added with polyelectrolytes during the layer by layer (LbL deposition ) . Polyelectrolyte deposited membranes can be a substrate for a thin film composite coating , e . g . , a polyamide coating that will result in a RO / FO / NF membrane .

[0221] EXAMPLES

[0222] Preparing amphipol-stabilized transmembrane AHMP formulation in two steps :

[0223] Pure Aquaporin Z (AqpZ ) in LDAO detergent is transferred into amphipol A8-35 by mixing the protein stock solution in LDAO with the amphipol , incubating them with defined time , and then removing the detergent . It is essential to add enough amphipol to achieve full reconstitution of all protein . Adding insufficient amphipol results in precipitation and loss of the protein . A two-step and a one-step method for purifying the amphipol stabilized protein were developed to .

[0224] First , the amphipol is dissolved overnight in water to ensure full solubilization . The solution is prepared at the maximum soluble concentration of the amphipol ( 200mg / ml for A8 -35 ) .

[0225] Next , the amphipol is mixed with the protein at a defined ratio , as much as 2 . 2mg amphipol per mg protein, or as little as 1 . Img amphipol per mg protein . The protein-amphipol mixture is incubated with shaking at 4 ° C for 2 hours . After incubation, purification is started immediately .

[0226] For the two step process , the mixture is first purified of detergent by a detergent removal spin filtration column (ThermoFischer item #87779 ) . This removes most of the LDAO present in the protein stock solution . After this step , a second step is used to remove any residual contaminants and deposit the AHMP in a defined buffer of IX PBS . This second step is a SEC purification using a Superdex200 10 / 300 GL column

[0227] Preparing AHMP formulation in one step

[0228] The one-step formulation production process differs from the two- step only in the purification step . All the same steps up to the purification step were followed for the one-step formulation as for the two-step formulation . For the one-step purification, the mixture of amphipol and protein is incubated with BioBeads SM-2 resin from BioRad at a ratio of 0 . 1 grams of BioBeads per ml of protein solution . The incubation is performed for 2 hours at RT with shaking , and the BioBeads are removed by filtration with 0 . 2um filter , yielding the final formulation .

[0229] Preparation of L-DOPA coating layer on porous support membrane Coatings were made according to the steps outlined below:

[0230] Dissolve Tris-HCl in RO water to a concentration of 0.158 % (w / w) and adjust the pH to 8.5

[0231] Dissolve L-DOPA powder in Tris-HCl buffer to a concentration of 0.2 % (w / w) and adjust to pH to 8.5

[0232] Insert porous support membrane into plastic round frames e.g. 23cm diameter

[0233] Put L-DOPA solution into glass containers and let membrane inserted into plastic frames get into contact with L-DOPA solution for 2hrs while glass container is continuously rotating at 33 rpm.

[0234] After 2hrs of contact time, take out the frames from L-DOPA solution and rinse the surface with RO water

[0235] Change L-DOPA solution in glass container with RO water after cleaning the glass container with RO water to get rid of excess L- DOPA solution

[0236] Let the plastic frames be in contact with RO water for 30min while the glass container is continuously rotating at 33 rpm to remove unreacted L-DOPA onto membrane support.

[0237] Plastic frames disassembled and from L-DOPA coated support membrane 9 cm x 13 cm rectangles are cut for next step.

[0238] Preparation of activated L-DOPA surface for AHMP coupling

[0239] Below steps describe how L-DOPA coated surface is activated. lx PBS buffer is prepared using NaCl: 137 mM, KC1: 2.7 mM, Na2HPO4 :

[0240] 10 mM, KH2PO4: 1.8 M in RO water and pH is adjusted 5 by using HC1.

[0241] Dissolve EDC and NHS powders in lx PBS buffer pH 5 to concentrations of 0.058 % (w / v) and 0.076 % (w / v) , respectively to prepare activation solution and after powders are dissolved adjust the pH to 5.

[0242] Prepare a rotating platform that can hold 2 containers each inside fume hood.

[0243] Take activation solution to fume hood containing glass containers on the rotating table. Pour approximately 250ml of activation solution into each container.

[0244] Transfer 2-3 L-DOPA coated membrane pieces into the activation solution by placing L DOPA side down into each container. After adding all L-DOPA coated membranes, start at 35min time and activate rotation at 34 per min.

[0245] Preparation of AHMP coupled membrane with activated L-DOPA coating layer

[0246] Below steps describe how AHMP is coupled with activated L-DOPA coated porous membrane surface. lx PBS buffer is prepared using NaCl: 137 mM, KC1: 2.7 mM, Na2HPO4 : 10 mM, KH2PO4: 1.8 mM in RO water and pH is adjusted 8.5 by using NaOH.

[0247] AqpZ formulation with A8-35 amphipol is added into lx PBS solution at pH 8.5 at a concentration of 0.002 mg / ml.

[0248] 350ul Betamercaptoethanol (BME) is added into activation solution to quench the reaction. Glass container is mixed by swirling the container .

[0249] Membranes to be AHMP coupled are washed with lx PBS solution at pH 8.5 three times and then fastened into a rectangle mold to apply coupling solution and mold is fastened by paper clips .

[0250] Coupling solution is poured on the membrane and coupling reaction is performed for 45 minutes at RT while stationary. lx PBS buffer is prepared using NaCl: 137 mM, KC1: 2.7 mM, Na2HPO4 :

[0251] 10 mM, H2PO4 : 1.8 M in RO water at pH 7.4.

[0252] Pour the coupling solution and wash the AHMP coupled membrane surface with lx PBS solution at pH 7.4.

[0253] Preparation of L-DOPA / A8-35 : AqpZ formulation (one step) layer deposition on porous membrane support

[0254] Below steps describe how AHMP is entrapped on the porous membrane surface together with L-DOPA deposition.

[0255] Dissolve Tris-HCl in RO water to a concentration of 0.158 % (w / w) and adjust the pH to 8.5

[0256] Dissolve L-DOPA powder in Tris-HCl buffer to a concentration of 0.2 % (w / w) and adjust to pH to 8.5

[0257] Add AqpZ : A8-35 formulation at a concentration of 0.002mg / ml into L-DOPA solution in Tris-HCl buffer

[0258] Insert porous support membrane into plastic round frames e.g. 23cm diameter

[0259] Put L-DOPA-A8-35 : AqpZ solution (one step) into glass containers and let membrane inserted into plastic frames get into contact with one step solution for 2hrs while glass container is continuously rotating at 33 rpm.

[0260] After 2hrs of contact time, take out the frames from L-DOPA solution and rinse the surface with RO water

[0261] Change L-DOPA solution in glass container with RO water after cleaning the glass container with RO water to get rid of excess L- DOPA solution

[0262] Let the plastic frames be in contact with RO water for 30min while glass container is continuously rotating at 33 rpm to remove unreacted L-DOPA onto membrane support. Plastic frames disassembled and from L-DOPA coated support membrane 9 cm x 13 cm rectangles are cut for next step .

[0263] Preparation of handmade TFC RO membranes with AHMP charged onto support membranes in sequential manner

[0264] These membranes were made according to the steps outlined below :

[0265] Dissolve MPD in RO water to get a 3 % (w / w) concentration

[0266] Dissolve TMC in Isopar to a final concentration of 0 . 12 % (w / w )

[0267] AHMP charged membranes with sequential method are washed with 3x RO water prior to interfacial polymerization to get rid of PBS solution .

[0268] Membrane is loaded into a rectangle mold and l OmL MPD solution is poured into mold and react for 30sec and then excess MPD solution is removed by a roller .

[0269] After excess MPD removal , TMC solution is poured to react with MPD for 30sec and then excess TMC is poured into waste .

[0270] Membrane is dried until Isopar left the membrane surface , then membrane soaked into RO water .

[0271] Example 1 : Preparation of L-DOPA one step AHMP charging and preparation of polyamide membranes

[0272] Materials :

[0273] L-DOPA was purchased from Sigma Aldrich . Tris-HCl buffer was purchased from Sigma Aldrich, item # 10812846001 .

[0274] AHMP was prepared according to the one-step formulation described above .

[0275] Preparation : 0,4728 g Tris-HCl is dissolved in 299,527g RO water and pH is adjusted to 8.5.

[0276] 0, 6 g L-DOPA is dissolved in 298,49 g Tris-HCl buffer.

[0277] Let the solution mix for lOmin at 300 rpm and then adjust the pH by NaOH to 8.5.

[0278] Add AHMP sample at 0.91 g into pH adjusted L-DOPA solution,

[0279] Frame membrane plastic frames,

[0280] Pour 150mL L-DOPA solution into glass dish, insert membrane in glass dish, then put 150ml L-DOPA solution to the backside of the frame,

[0281] Rotate membranes continuously for 2 hrs at 33 rpm at RT,

[0282] Flush and replace solution with RO water on coated membrane, continue to shake for 30 min at 33 rpm at RT .

[0283] Cut 9 cm x 13 cm membrane area with a scalpel,

[0284] Dissolve 0.3 g MPD into 9.7 g RO water and mix it with 300rpm at room temperature and adjust pH to 8.

[0285] Dissolve 0,00864 g TMC into 7.19g Isopar E and mix it with 300 rpm speed at RT .

[0286] Insert AHMP charged support onto mold used for PA coating

[0287] Fasten mold with o-ring and plastic.

[0288] Pour 10 ml MPD solution into the mold and let it react with 30s,

[0289] Remove excess MPD by pouring it, then until no water droplets left MPD is rolled from membrane surface.

[0290] Insert AHMP charged membrane into mold again,

[0291] Pour 10 ml TMC solution and fasten mold,

[0292] Let solution react for 30s, then pour excess solution. Let Isopar dry out of the membranes, then insert membrane into RO water until testing.

[0293] Membrane is tested for 250 ppm NaCl solution at 4.5 bar after 72 hrs of compaction with at least 3 replicates.

[0294] Example 2 : Preparation of L-DOPA coating and preparation of polyamide membranes

[0295] Materials :

[0296] L-DOPA was purchased from Sigma Aldrich. Tris-HCl buffer was purchased from Sigma Aldrich, item # 10812846001.

[0297] Preparation :

[0298] 0,4728 g Tris-HCl is dissolved in 299,527g RO water and pH is adjusted to 8.5.

[0299] 0, 6 g L-DOPA is dissolved in 299,4 g Tris-HCl buffer.

[0300] Let the solution mix for lOmin at 300 rpm and then adjust the pH by NaOH to 8.5.

[0301] Frame membrane plastic frames,

[0302] Pour 150mL L-DOPA solution into glass dish, insert membrane in glass dish, then put 150ml L-DOPA solution to the backside of the frame,

[0303] Rotate membranes continuously for 2 hrs at 33 rpm at RT,

[0304] Flush and replace solution with RO water on coated membrane, continue to shake for 30 min at 33 rpm at RT .

[0305] Cut 9 cm x 13 cm membrane area with a scalpel,

[0306] Dissolve 0.3 g MPD into 9.7 g RO water and mix it with 300rpm at room temperature and adjust pH to 8. Dissolve 0,00864 g TMC into 7.19g Isopar E and mix it with 300 rpm speed at RT .

[0307] Insert AHMP charged support onto mold used for PA coating

[0308] Fasten mold with o-ring and plastic

[0309] Pour 10 ml MPD solution into the mold and let it react with 30s,

[0310] Remove excess MPD by pouring it, then until no water droplets left MPD is rolled from membrane surface

[0311] Insert AHMP charged membrane into mold again,

[0312] Pour 10 ml TMC solution and fasten mold,

[0313] Let solution react for 30s, then pour excess solution

[0314] Let Isopar dry out of the membranes, then insert membrane into RO water until testing

[0315] Membrane is tested for 250 ppm NaCl solution at 4.5 bar after 72 hrs of compaction with at least 3 replicates.

[0316] Example 3: Preparation of Dopamine Hydrochloride one step AHMP charging and preparation of polyamide membranes

[0317] Materials :

[0318] Dopamine hydrochloride was purchased from Sigma Aldrich. Tris-HCl buffer was purchased from Sigma Aldrich, item # 10812846001.

[0319] AHMP was prepared according to the one-step formulation described above .

[0320] Preparation :

[0321] 0,4728 g Tris-HCl is dissolved in 299,527g RO water and pH is adjusted to 8.5.

[0322] 0, 6 g Dopamine hydrochloride is dissolved in 298,49 g Tris-HCl buffer . Let the solution mix for lOmin at 300 rpm and then adjust the pH by NaOH to 8.5.

[0323] Add AHMP sample at 0.91 g into pH adjusted Dopamine hydrochloride solution,

[0324] Frame membrane plastic frames,

[0325] Pour 150mL Dopamine hydrochloride solution into glass dish, insert membrane in glass dish, then put 150ml Dopamine hydrochloride solution to the backside of the frame,

[0326] Rotate membranes continuously for 2 hrs at 33 rpm at RT,

[0327] Flush and replace solution with RO water on coated membrane, continue to shake for 30 min at 33 rpm at RT .

[0328] Cut 9 cm x 13 cm membrane area with a scalpel,

[0329] Dissolve 0.3 g MPD into 9.7 g RO water and mix it with 300rpm at room temperature and adjust pH to 8.

[0330] Dissolve 0,00864 g TMC into 7.19g Isopar E and mix it with 300 rpm speed at RT .

[0331] Insert AHMP charged support onto mold used for PA coating

[0332] Fasten mold with o-ring and plastic

[0333] Pour 10 ml MPD solution into the mold and let it react with 30s,

[0334] Remove excess MPD by pouring it, then until no water droplets left MPD is rolled from membrane surface

[0335] Insert AHMP charged membrane into mold again,

[0336] Pour 10 ml TMC solution and fasten mold,

[0337] Let solution react for 30s, then pour excess solution

[0338] Let Isopar dry out of the membranes, then insert membrane into RO water until testing Membrane is tested for 250 ppm NaCl solution at 4.5 bar after 72 hrs of compaction with at least 3 replicates.

[0339] Example 4 : Preparation of Dopamine Hydrochloride coating and preparation of polyamide membranes

[0340] Materials :

[0341] Dopamine hydrochloride was purchased from Sigma Aldrich. Tris-HCl buffer was purchased from Sigma Aldrich, item # 10812846001.

[0342] Preparation :

[0343] 0,4728 g Tris-HCl is dissolved in 299,527g RO water and pH is adjusted to 8.5.

[0344] 0, 6 g Dopamine hydrochloride is dissolved in 299,4 g Tris-HCl buffer .

[0345] Let the solution mix for lOmin at 300 rpm and then adjust the pH by NaOH to 8.5.

[0346] Frame membrane plastic frames,

[0347] Pour 150mL Dopamine hydrochloride solution into glass dish, insert membrane in glass dish, then put 150ml Dopamine hydrochloride solution to the backside of the frame,

[0348] Rotate membranes continuously for 2 hrs at 33 rpm at RT,

[0349] Flush and replace solution with RO water on coated membrane, continue to shake for 30 min at 33 rpm at RT .

[0350] Cut 9 cm x 13 cm membrane area with a scalpel,

[0351] Dissolve 0.3 g MPD into 9.7 g RO water and mix it with 300rpm at room temperature and adjust pH to 8.

[0352] Dissolve 0,00864 g TMC into 7.19g Isopar E and mix it with 300 rpm speed at RT . Insert AHMP charged support onto mold used for PA coating

[0353] Fasten mold with o-ring and plastic

[0354] Pour 10 ml MPD solution into the mold and let it react with 30s,

[0355] Remove excess MPD by pouring it, then until no water droplets left MPD is rolled from membrane surface

[0356] Insert AHMP charged membrane into mold again,

[0357] Pour 10 ml TMC solution and fasten mold,

[0358] Let solution react for 30s, then pour excess solution

[0359] Let Isopar dry out of the membranes, then insert membrane into RO water until testing

[0360] Membrane is tested for 250 ppm NaCl solution at 4.5 bar after 72 hrs of compaction with at least 3 replicates.

[0361] Example 5: Preparation of L-DOPA coating, sequential AHMP charging and polyamide membrane

[0362] L-DOPA was purchased from Sigma Aldrich. Tris-HCl buffer was purchased from Sigma Aldrich, item # 10812846001. PBS powder was purchased from Sigma Aldrich

[0363] Preparation :

[0364] 0,4728 g Tris-HCl is dissolved in 299,527g RO water and pH is adjusted to 8.5.

[0365] 0, 6 g L-DOPA is dissolved in 299,4 g Tris-HCl buffer.

[0366] Let the solution mix for lOmin at 300 rpm and then adjust the pH by NaOH to 8.5.

[0367] Frame membrane plastic frames, Pour 150mL L-DOPA solution into glass dish, insert membrane in glass dish, then put 150ml L-DOPA solution to the backside of the frame,

[0368] Rotate membranes continuously for 2 hrs at 33 rpm at RT,

[0369] Flush and replace solution with RO water on coated membrane, continue to shake for 30 min at 33 rpm at RT .

[0370] Cut 9 cm x 13 cm membrane area with a scalpel, lx PBS buffer is prepared by dissolving lx PBS powder in IL RO water and pH is adjusted 5 by using HC1.

[0371] Dissolve 0.29g EDC and 0.3895g NHS powders in lx PBS buffer pH 5 and fill volume up to 500mL and dissolve them by mixing a 300 rpm.

[0372] Rotating platform is prepared and glass dish is put inside the fumehood .

[0373] Take activation solution to fume hood containing glass containers on the rotating table. Pour approximately 250ml of activation solution into the glass dish.

[0374] Transfer 2-3 L-DOPA coated membrane pieces into the activation solution by placing L DOPA side down into each container. After adding all L-DOPA coated membranes, start at 35min time and activate rotation at 34 per min at RT .

[0375] IL lx PBS buffer is prepared by dissolving lx PBS powder in IL RO water and pH is adjusted 8.5 by using NaOH.

[0376] 0,48 ml AqpZ formulation with A8-35 amphipol is added into 14,52 ml lx PBS solution at pH 8.5 at a concentration of 0.002 mg / ml.

[0377] 350ul Betamercaptoethanol (BME) is added into activation solution to quench the reaction. Glass container is mixed by swirling the container . Membranes to be AHMP coupled are washed with 200mL lx PBS solution at pH 8 . 5 three times and then fastened into a rectangle mold to apply coupling solution and mold is fastened by paper clips .

[0378] 15 ml Coupling solution is poured on the membrane and coupling reaction is performed for 45 minutes at RT while molds are stationary .

[0379] IL lx PBS buffer is prepared by dissolving lx PBS powder in IL RO water at pH 7 . 4 .

[0380] Pour the coupling solution and wash the AHMP coupled membrane surface with 15 ml lx PBS solution at pH 7 . 4 .

[0381] Store membranes in 50mL PBS solution at pH 7 . 4 until they are PA coated . Exchange PBS buffer with RO water before continue ,

[0382] Dissolve 0 . 3 g MPD into 9 . 7 g RO water and mix it with 300rpm at room temperature and adj ust pH to 8 .

[0383] Dissolve 0 , 00864 g TMC into 7 . 19g Isopar E and mix it with 300 rpm speed at RT .

[0384] Insert AHMP charged support onto mold used for PA coating

[0385] Fasten mold with o-ring and plastic

[0386] Pour 10 ml MPD solution into the mold and let it react with 30s ,

[0387] Remove excess MPD by pouring it , then until no water droplets left MPD is rolled from membrane surface

[0388] Insert AHMP charged membrane into mold again,

[0389] Pour 10 ml TMC solution and fasten mold,

[0390] Let solution react for 30s , then pour excess solution

[0391] Let Isopar dry out of the membranes , then insert membrane into RO water until testing

[0392] Membrane is tested for 250 ppm NaCl solution at 4 . 5 bar after 72 hrs of compaction with at least 3 replicates . Example 6: Fluorescent amphipol preparation protocol

[0393] A8-35 (commercial, 150 mg) was dissolved in 6 mL of xO .05 PBS (pH = 6.81, pH adjusted with HC1) . Fluoresceinamine (24.1 mg) was dissolved in 1.5 mL DMSO and injected into the aqueous mixture of A8-35. Then, the mixture was supplemented with 6 mL of x0.05 PBS (pH = 6.81) and 1.5 mL of EDC / NHS solution (x0.05 PBS, pH = 6.81) . EDC / NHS amounts were 53.9 and 16.0 mg, respectively. The mixtures were left to stir overnight (20-24 h) in 20 mL vial under ambient temperature. The vial was fully covered with aluminum foil during the time of the reaction.

[0394] The content of the vial is then transferred to a dialysis bag with MWCO of 3.5 kDa. The contents are dialyzed against MiliQ (1.8-2.0 L) for 3 days. After that, the dialysate is exchanged with fresh MiliQ (1.8-2.0 L) and the contents are dialyzed for 1 day. Finally, the samples are freeze-dried, yielding a yellow, light powder.

[0395] Example 7 : Labelling of protein amines with NHS esters and purification .

[0396] Day 0 — Column Preparation

[0397] Prepare 1 L of lx PBS (pH 7.4) and filter (0.2 pm) .

[0398] Equilibrate column: flush with Milli-Q water (3 CVs) followed by lx PBS (3 CVs) .

[0399] Allow the column to equilibrate overnight in PBS.

[0400] Day 1 — Protein Labelling

[0401] Prepare NHS-ester stock (e.g., Atto dye) in dry DMSO; thaw if premade .

[0402] Gently mix AqpZ protein, check for precipitation, and take SDS sample #1 (input) .

[0403] Concentrate protein (< 4.5 mL) using a 30 kDa spin filter. Combine concentrated protein with the NHS ester according to the AiG2 reaction table.

[0404] Incubate 1 h at room temperature on a tilting mixer; then take SDS sample #2 (post-reaction) . Day 2 — Purification

[0405] Equilibrate Superdex 200 PG 16 / 600 column in lx PBS.

[0406] Load reaction mixture and purify using 1 mL / min flow rate.

[0407] Collect fractions showing L 100 mAU absorbance; pool these as the labelled protein fraction.

Claims

65CLAIMS1 . A semi-permeable membrane comprising a porous support and a protein assembly comprising an amphiphilic heteropolymer and a membrane protein wherein the semi-permeable membrane comprises a first layer consisting of a polyamide thin film composite layer .2 . The semi-permeable membrane according to claim 1 , wherein the porous support comprises a second layer, which is a matrix of at least one self-oligomerised or self-polymerised compound or a mixture thereof forming a layer on said porous support .3 . The semi-permeable membrane according to either one of claims 1 or 2 , wherein at least some of the polyamide thin film composite layer is coating the protein assembly .4 . The semi -permeable membrane according to any one of claims 1 to 3 , wherein the water permeability of said semi -permeable membrane is at least 18L / (m2 * h *bar ) and salt rej ection is at least 99% at 500ppm NaCl , 4 . 9 bar , 25 ° C, pH 6-8 .5 . The semi-permeable membrane according to any one of claims 1 to 4 , wherein the membrane protein is a membrane water channel , a membrane ion channel , or a membrane transporter .6 . The semi-permeable membrane according to any one of claims 1 to 5 , wherein the amphiphilic heteropolymer is an amphipol .7 . The semi-permeable membrane according to any one of claims 1 to 6 , wherein the amphiphilic heteropolymer is not a block co-polymer .8 . The semi-permeable membrane according to any one of claims 1 to 7 , wherein the second layer formed by a matrix of a self- oligomerised or self-polymerised compound layered on said porous support comprises poly ( L-3 , 4-dihydroxyphenylalanine ) , poly ( L-DOPA) .669 . A method of forming a semi-permeable membrane , the method comprisin :- providing a porous support ,- stabilising a transmembrane protein with an amphiphilic heteropolymer to form an amphiphilic heteropolymer- stabilised transmembrane protein assembly, and- incorporating the amphiphilic heteropolymer-stabilised transmembrane protein assembly onto the semi -permeable membrane .- forming a first layer comprising polyamide on the semi- permeable membrane by interfacial polymerisation of an amine and an acyl halide .10 . The method according to claim 9 , further comprising the step of immobilising the amphiphilic heteropolymer-stabilised membrane protein assembly within the structure of the semi- permeable membrane .11 . The method according to either one of claims 9 or 10 , further comprising forming a second layer on the porous support by contacting a self-oligomerisable or self- polymerisable compound with a surface of the porous support to form a matrix .12 . The method according to claim 11 , wherein the self- polymerisable compound is L-3 , 4-dihydroxyphenylalanine (L- DOPA) .13 . The method according to any one of claims 10 to 12 , wherein EDC-NHS chemistry is used to immobilize the heteropolymer-stabilised membrane protein assembly onto the semi-permeable membrane .14 . The method according to any one of claims 11 to 13 , wherein at least some of the amphiphilic heteropolymer- stabilised membrane protein assembly is included in a solution comprising the self-oligomerisable or self-polymerisable compound to be contacted on the porous support .6715 . The method according to any one of claims 11 to 14 , wherein the heteropolymer-stabilised membrane protein assembly immobilisation is achieved through covalent linking of one or more membrane protein amino acid ( s ) primary amines and a carboxylic acid group present on the porous support or on the self-oligomerized or self-polymerized compound in the matri .

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